Generation and Characterization of B72.3 - Cancer Research

(CANCER RESEARCH 48.4588-4596, August 15, 1988]

Generation and Characterization of B72.3 Second Generation MonoclonalAntibodies Reactive with the Tumor-associated Glycoprotein 72 Antigen

Raffaella Muraro, Masahide Kuroki, David Wunderlich, Diane J. Poole, David Colcher, Ann Thor, John W. Greiner,Jean F. Simpson, Alfredo Molinolo, Philip Noguchi, and Jeffrey Schlom1

Laboratory of Tumor Immunologi' and Biology, National Cancer Institute, NIH[R. M., M. K., D. W., D. J. P., D. C., A. T., J. W. G., J. F. S., A. M., J. S.J, and Officeof Biologies, National Center for Drugs and Biologies, Food and Drug Administration [P. N.], Bethesda, MD 20892

ABSTRACT

Monoclonal antibody (MAb) B72.3 was generated using a membrane-enriched fraction of a human mammary carcinoma biopsy. It has demonstrated reactivity to the majority of human adenocarcinomas includingcolorÃ©ela!,gastric, pancreatic, ovarian, endometrial, mammary, and non-small cell lung cancer and no or weak reactivity to normal adult tissues,with the exception of secretory endometrium. The B72.3-reactive antigen,termed tumor-associated glycoprotein (TAG)-72, has been puntini andused as an Â¡mmunogento generate B72.3 second generation MAbs. Sincethe source of purified TAG-72 was a human colon cancer (CC) \enograft,these MAbs have been given a CC designation. Twenty-eight CC MAbs,all immunoglobulin Gs, have been generated and shown to be reactivewith TAG-72 and via both radioimmunoassay and immunohistochemicalanalyses show differential reactivity to carcinoma versus normal adulttissue biopsies. Nine CC MAbs Â«Ml. 15, 29, 30, 40, 46, 49, 83, and92) were selected for further characterization. As a result of analysesusing direct-binding radioimmunoassay to a range of human carcinomas,Western blotting, live cell surface binding assays, five liquid competitionradioimmunoassays, and A,, measurements, all nine CC MAbs could bedistinguished from each other and from B72.3. The K, of B72.3 wasdetermined to be 2.54 x id1' M '; all the CC MAbs demonstrated higher

K.S with MAbs CC92, 49, and 83 having K.s of 14.26, 16.18, and 27.72x 10' M"', respectively. These studies thus demonstrate that one or more

of the anti-TAG-72 CC MAbs may be more efficient than B72.3, oruseful in combination with B72.3, toward the further study of humancarcinoma cell population and the diagnostic and therapeutic procedurespresently utilizing MAb B72.3.

INTRODUCTION

MAb2 B72.3 was generated by the immunization of mice witha membrane-enriched fraction of a human breast carcinomabiopsy (1). B72.3 has been shown to be reactive with a widerange of human carcinomas including colorectal, gastric, pancreatic, mammary, ovarian, and endometrial carcinomas andadenocarcinomas of the lung (2-7). B72.3 has also been shownto be nonreactive or only weakly reactive to a wide range ofnormal adult tissues (4) with the exception of secretory endometrium; however, proliferative and resting endometria (ofpostmenopausal women) were nonreactive with B72.3 (8). TheB72.3-reactive antigen, TAG-72, is expressed at high levels incertain human fetal tissues including fetal intestine (4).

TAG-72 has been purified from the LS-174T human coloncancer xenograft via several Chromatographie procedures, including sequential passages through B72.3 affinity columns.These studies and the characterization of the TAG-72 antigen

Received 2/24/88; revised 5/12/88; accepted 5/20/88.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 I'.S.C. Section 1734 solely to indicate this fact.

1To whom requests for reprints should be addressed at Laboratory of Tumor

Immunology and Biology. National Cancer Institute. Building 10. Room 8B07.Bethesda. MD 20892.

2The abbreviations used are: MAb. monoclonal antibody; TAG-72, tumor-

associated glycoprotein 72; RIA. radioimmunoassay; CEA, carcinoembryonicantigen: BSA, bovine serum albumin: PBS. phosphate-buffered saline; CC. coloncancer; SDS-PAGE. sodium dodecyl sulfate-polyacrylamide gel electrophoresis;TBS, Tris-buffcred saline.

have been detailed elsewhere (9). TAG-72 can be characterizedas a high molecular weight glycoprotein with characteristics ofa mucin. There have been numerous studies recently on tumor-associated mucin glycoproteins (10-20). The TAG-72 antigenhas been shown to be distinct biochemically, immunologically,and in terms of expression from CEA and the antigens detectedby other anti-tumor MAbs such as 19-9, 17-1A, OC 125,DUPAN2, the anti-human milk fat globule membrane MAbs,

and DF3 (6, 7, 21, 22).Although MAb B72.3 is reactive with the majority of human

carcinoma biopsies, it has been shown to be nonreactive or verypoorly reactive with the vast majority of human carcinoma linesin culture (23). The LS-174T colon carcinoma cell line was 1of 2 of over 40 cell lines tested for B72.3 reactivity that waspositive. Studies have shown that several parameters, includingspatial configuration, affect the expression of the TAG-72antigen (23, 24). The recent studies (8) demonstrating thatTAG-72 is expressed only in secretory phase endometriumindicate that hormones may also be involved in TAG-72 expression. As with most MAbs to human carcinoma antigens, a greatdeal of antigenic heterogeneity has also been detected usingMAb B72.3 in carcinoma masses from the same or differentpatients or even within a given carcinoma mass (see Ref. 21 forreview).

There are several aspects of the management of humancarcinoma for which MAb B72.3 has potential utility. A serumassay has been developed using B72.3 in which approximately50% of carcinoma patients' sera have been shown to contain

TAG-72 as compared to approximately 4% of the controlpopulation (25). This assay has been shown to be distinct fromother serum assays currently being used for carcinoma detection(26). B72.3 has also been used in numerous studies to detectcarcinoma in human effusions (27, 28) and fine needle aspiration biopsies (5, 29, 30) and as a diagnostie tool to distinguishbetween adenocarcinoma of the lung and (a) non-small celllung cancer (5) or (b) malignant mesothelioma (31).

Recent clinical studies using '-"I-labeled B72.3 IgG have

demonstrated specific MAb localization of carcinoma lesionsvia direct biopsy analysis in approximately 70% of colorectaland ovarian carcinoma patients (32-34). Studies using dual-label B72.3 ("'I-labeled B72.3 IgG injected i.p. simultaneouslywith '"I-labeled B72.3 injected i.v.) in individual patients have

shown that the i.p. administered B72.3 will localize carcinomaperitoneal implants with greater efficiency (35). Gamma scansof patients receiving '-"I-labeled B72.3 i.p. could detect lesions

that were not detectable using computer-assisted tomographicscan or X-ray in approximately 20-30% of cases. Many tumor

lesions localizing both i.v. and i.p. administered B72.3 did soat ratios of 5:1 to over 50:1 versus normal tissues. An intraoperative hand-held gamma probe used subsequent to radiola-

beled B72.3 administration has shown localization of B72.3 in70-80% of carcinoma lesions with the ability to detect occultcarcinoma intraoperatively with the probe in approximately

4588

Research. on January 3, 2019. © 1988 American Association for Cancercancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

SECOND GENERATION MAbs TO TAG-72

20% of patients (36, 37). Therapy trials using radiolabeledB72.3 are now in progress.

In view of the potential clinical applications of MAb B72.3and our ability to purify the reactive TAG-72 antigen, thedecision was made to prepare "second generation" anti-TAG-

72 MAbs. This was done with the expectation that there wasno a priori reason for B72.3 to be the ideal anti-TAG-72 MAb.With the selective advantage of a purified antigen at hand,numerous assays were used to select those MAbs that werereactive with TAG-72 but distinct from B72.3, so that theycould be used as a more efficient substitute for, or in combination with, B72.3 in subsequent applications. We describe herethe generation and characterization of 28 anti-TAG-72 MAbswith a more comprehensive analysis of nine of these secondgeneration MAbs.

MATERIALS AND METHODS

Source of Cell Lines and Tissues and Tumor Preparation. Neoplasticand normal human adult tissues were obtained from the Departmentsof Surgery and Pathology of the George Washington University, Washington, DC.

LS-174T (38), a human colon adenocarcinoma cell line, and twonormal human fibroblast lines, WI-38 and MRC-5, were obtained fromthe American Type Culture Collection (Rockville, MD). The LS-174Tcells were grown in Eagle's minimal essential medium with nonessential

amino acids, supplemented with 10% fetal calf serum and gentamicin(50 Mg/m') while both fÃbroblastcell lines were routinely maintained inEagle's basal medium (Eagle's salts) supplemented with Ix glutamine,

50 Mg/ml gentamicin, and 10% heat-inactivated fetal bovine serum. TheFlow 4000 fibroblast cell line was obtained from Flow Laboratories(Rockville, MD) and was grown in Dulbecco's modified Eagle's medium

supplemented with gentamicin and 10% heat-inactivated fetal bovineserum. All lines were subpassaged at a 1:5 ratio weekly.

Preparation of Immunogen. TAG-72 was purified from xenograftsgenerated from the LS-174T cell line by a modification of the methodsof Johnson et al. (9). Approximately 3 g of frozen LS-174T humancarcinoma xenograft were homogenized with an Omni-Mixer for 45 sin 20 mM Tris-HCl (pH 7.2) and 150 mM NaCl (TBS). The homogenized xenograft was then filtered through glass wool and loaded ontoa Sepharose CL-4B column-sizing column (5.5 x 25 cm; Pharmacia,Uppsala, Sweden) that was previously equilibrated in TBS. The columnwas eluted using TBS and 7.0-ml fractions were collected and tested ina solid-phase RIA (see below) to localize the TAG-72. Peak fractionswere pooled and loaded onto a B72.3 affinity column [100 ml Reactor-Gel MW65F (Pierce, Rockford, IL) coupled with 200 mg of B72.3IgG] equilibrated with TBS. The column was washed with TBS, andthe bound protein was eluted with 3.0 M Nal in TBS. Fractions (5 ml)were collected and tested in a solid-phase RIA to localize the TAG-72.Peak fractions were pooled, dialyzed against 20 mM Tris-HCI (pH 7.2),concentrated using Aquacide II (Calbiochem, San Diego, CA), andredialyzed against TBS. The purified TAG-72 was stored at â€”70Â°C

until used as immunogen or antigen in subsequent RIAs.Preparation of Hybridomas. Three 4-week-old BALB/c mice were

immunized by i.p. inoculation of 10 MgTAG-72, purified as describedabove, which had been premixed with an equal volume of completeFreund's adjuvant. After 80 days, the mice received booster doses of 50

Mgof purified TAG-72 premixed with an equal volume of incompleteFreund's adjuvant. Seven days later the mice received i.v. 10 Mg of

purified TAG-72 in saline. Spleens were harvested 3 days later for cellfusion.

Hybridomas were prepared using a modification of the method ofHerzenberg et ai. (39). Single cell suspensions of spleen cells from theimmunized mice were made by passing the spleen tissue of the micethrough a No. 3 mesh stainless steel screen. The spleen cells and NS-1mouse myeloma cells (ATCC No. TIB-18) were washed in RPMI 1640medium, containing 2.0 mM glutamine, 1.0 mM sodium pyruvate, 50units/ml penicillin. 50 Mg/ml streptomycin, and 0.25 Mg Fungizone,

mixed at a 4:1 ratio, and fused with 50% (v/v) polyethylene glycol (A/,1500) (BDH Chemical, Ltd., Poole, England). After fusion, individualwells of 96-well microtiter plates (Costar, Cambridge, MA) were seededwith 1 x IO6total cells (0.1 ml) of the cell suspension. Fused cells werethen selected for growth with hypoxanthine-aminopterin-thymidinemedium. Hybridomas were screened for desired reactivity by using asolid-phase RIA, and selected wells were cloned twice by limitingdilution.

Preparation of Extracts. Tissue extracts were prepared by a modification of a method used by Colcher et al. (40). Tissues were homogenized for 2 min in a homogenizer at top speed (Tekmar, Cincinnati,OH) and then subjected to nitrogen pressure at 1000 psi (Parr Instrument Co., Moline, IL). The homogenate was centrifuged for 15 min at1000 x g, and the resultant supernatant was sonicated (Heat SystemsUltrasonics, Plainview, NY) for 1 min at the maximum setting. Aftersonication, the homogenate was further clarified by centrifugation at10,000 x g for 15 min, and the supernatant protein content wasdetermined by the method of Lowry et al. (41).

Solid-Phase Radioimmunoassays. Hybridoma supernatants were assayed for specific antibody production in solid-phase RIA using cellextracts from different colon carcinomas, normal tissues, purified TAG-

72, and purified CEA. Fifty M' containing 5 Mgof cell extract, 5 unitsof TAG-72, or 20 ng of purified CEA were used in solid phase RIAs asdescribed previously (23).

To determine the isotype of the different CC MAbs a RIA wasdeveloped using 100 ng of an F(ab')2 fragment of a sheep anti-murineIgG F(ab')2 fragment dried on each well (Jackson ImmunoResearch,

West Grove, PA). The assay was run as described (23) using 50 n\ ofhybridoma tissue culture supernatant fluid per well incubated at 37Â°C

for 1 h. The plates were washed with PBS containing 1% BSA and anÂ¡sonpc specific rabbit anti-mouse immunoglobulin antiserum (1:2000dilution; Organon Teknika, Durham, NC) was added for l h at 37Â°Cfollowed by 125I-protein A for l h additional at 37Â°C.

Additional antibodies were used as controls in the solid phase RIAs,as well as immunoperoxidase studies. Anti-CEA MAbs Bl.l and COL-1 have been described previously (42, 43). MOPC-21 (IgGl) (44, 45)was used as a negative control (Organon Teknika) for binding toextracts in all solid phase RIAs, and in all cases (Tables 1 and 2) gaveless than 500 cpm bound per well.

Immunoperoxidase Studies. Reactivity of hybridoma tissue culturesupernatants with formalin-fixed or frozen 5-Mm tissue sections wasdetermined using a modification of the avidin-biotin-peroxidase complex (Vectastain ABC kit; Vector Laboratories, Inc., Burlingame, CA)method (4). For each CC MAb tested, isotype identical control antibodies (MOPC-21 or UPC-10) (44, 45) were used at identical concentrations and conditions on parallel sections; no reactivity was observedwith the control antibodies.

Live-Cell Radioimmunoassay. Live-cell RIAs utilizing established celllines were performed by a modification of the method of Horan Handet al. (23).

Flow Cytometry. Flow cytometric analysis of human carcinoma cellsisolated from a pleura! effusion of a patient diagnosed with metastaticbreast carcinoma (Drs. James McCoy and Ruth Cannon, Immunoquest,Inc., Rockville, MD) was performed as described previously (46). Thecellular component of the pleural effusion was initially pelleted bycentrifugation at 800 x g for 5-10 min. The RBC were lysed in ahypotonie NH4C1 solution and the remaining tumor cells were isolatedby differential centrifugation on a Ficoll-Hypaque gradient (LSM, 6.2%Ficoll; Organon Teknika Corp.). Analysis of the human carcinoma cellswas carried out using an Ortho Cytofluorograf system 50H (OrthoPharmaceutical Corp., Raritan, NJ) with a blue laser excitation of 200mW at 488 nm.

Western Blotting Analyses. Cell extracts diluted in SDS-PAGE sample buffer [0.125 M Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, and10% /3-mercaptoethanol] were analyzed using 3-12% linear gradientSDS-PAGE. After electrophoresis, proteins were transferred to nitrocellulose paper [0.45-Mm pore size) at 4Â°Cfor 4 h at 30 V in transfer

buffer [25 HIM Tris-HCl (pH 8.3), 192 mM glycine, and 20%methanol). The blots were then incubated in PBS containing 5% BSAfor l h at room temperature and washed with PBS containing 0.05%

4589




Tween 20. Ten ml of each purified antibody were added at a concentration of 10 ng/ml and incubations were continued for 2 h at roomtemperature with gentle agitation. After washing with PBS containing0.05% Tween 20, the blots were incubated for l h at room temperaturewith 5 x IO5cpm/ml '"I-labeled goat anti-mouse IgG. The filters werethen washed extensively overnight and exposed to Kodak XAR X-rayfilm with an intensifying screen at -70T for 3 or 13.5 h. For allexperiments, purified MOPC-21 IgG was used as a negative control.

Purification of MAb IgG. MAb IgG was purified from ascitic fluidby ammonium sulfate precipitation followed by ion-exchange chromatography either by open-column methodology on DE-52 (Whatman,Clifton, NJ), by high-performance liquid chromatography on DEAE-5PW (Waters, Milford, MA), or on Bakerbond ABx columns (J. T.Baker Chemical Co., Philipsburg, NJ). The antibodies were eluted by a10-100 mM sodium phosphate (pH 7.5) gradient, a gradient of 20 mMTris-HCl (pH 8.5) to 300 mM NaCl in 20 mM Tris-HCl (pH 7.0), or25mM 2-(A'-morpholino)ethanesulfonate (pH 5.6) to l M sodiumacetate (pH 7.0). Fractions were analyzed by SDS-PAGE, and appropriate fractions were pooled. Purified IgG was dialyzed against PBSand quantitated by determining protein concentration by the methodof Lowry et al. (41).

Competition RIA. Antibody samples were serially diluted in 1% BSAin PBS and added (in 25 Â»illto plates in triplicate, containing l Â¿tg/wellof the LS-174T tumor extract. Following a 6-h incubation at 4Â°C,25nl of '"I-labeled MAb (50,000 cpm) were added to each well and

incubated for an additional 18 h at 4 ( . The plates were then washedwith 1% BSA in PBS. The bound '25I-labeied MAb was measured in a

gamma counter. The percentage of inhibition, as compared to a controlbuffer sample, was determined.

Determination of Affinity Constant (A'Â«).The affinity constants of the

CC MAbs to TAG-72 were determined by a solid phase immunoassayaccording to a modification of Heyman et al. (47). Thirty jil of purifiedTAG-72 (8.4 units) diluted in PBS (pH 7.2) were dried down in 96-well polyvinyl chloride microtiter plates. Any remaining nonspecificactive groups were blocked with 5% BSA in PBS. Twenty ^1 of 1:1.5serial dilutions of each purified antibody starting at 1 Mg/ml were addedto the wells. After incubation overnight at 4Â°C,the plates were washed3 times with PBS. 12M-labeledgoat anti-mouse IgG (75,000 cpm/25 M')was added and left to react for l h at 37Â°C.After 3 washings, the

individual wells were counted. In order to convert cpm values toconcentration of bound antibody, the remaining free antibody in thesupernatant that had been incubated with the antigen was incubated onanother plate precoated with sheep anti-mouse IgG at a concentrationof 4 ng/ml and detected with '"I-labeled goat anti-mouse IgG. Then

the concentration at which no antibody was left in the supernatant wasestimated for each antibody and utilized to convert cpm values toconcentration of bound antibody. To calculate K,, the method ofScatchard (48) was used.

RESULTS

Analyses of Hybridoma Cultures. Three mice were immunizedwith purified TAG-72, as described in "Materials and Methods." Using methodology described previously (43), the super

natant fluids of 2567 fusion culture wells (96 cm) were testedwith solid-phase RIA for reactivity to a breast cancer biopsyextract and for lack of reactivity to identically prepared membrane-enriched extracts of biopsies of a normal human liverand spleen; 433 cultures contained supernatant fluid that metthese criteria. These 433 cultures were then passaged to 2-cmwells and tested with solid-phase RIA for reactivity to a breastcancer extract and a colon carcinoma biopsy extract and forlack of reactivity to biopsy extracts of the following normaltissues: kidney, bone marrow, lung, liver, colon, stomach, thyroid, polymorphonuclear leukocytes, and erythrocytes. Ninety-nine cultures were positive for reactivity with the two carcinomaextracts and negative for reactivity with the nine normal tissues.Cells from these 99 cultures were cloned by end-point dilution

into 9504 wells (96 wells per original culture), and each culturewas checked for the presence of single cells. Cultures grown upfrom single cell clones were checked with solid-phase RIA forthe production of immunoglobulins that were reactive with acolon cancer extract and negative for normal liver. Following asecond cloning as described above, 28 hybridoma cultures (double cloned) were selected on the basis of (a) intensity of reactivity with purified TAG-72 and a colon cancer extract and forlack of reactivity to normal liver extract and CEA and (b)reactivity with sections of formalin-fixed tissues using the im-munoperoxidase method (see below). These 28 hybridomas, allfrom different original cultures in 96-cm wells, have been designated CC MAbs since the immunogen was from the LS-174Tcolon cancer (CC) xenograft, while the immunogen for theoriginal MAb B72.3 was a breast cancer biopsy.

The CC MAbs were isotyped using solid-phase RIAs asdescribed in "Materials and Methods." Since the goat anti-

mouse IgG (light chain-specific) reagent used in screening thehybridomas was selective for reactivity with the murine IgGisotype, it is not surprising that all the CC MAbs were IgG.MAb CC60 was an IgG2a, MAbs CCI5 and 48 were IgG2b,and the rest were IgGl (Table 1).

Solid-Phase Radioimmunoassays. The 28 CC MAbs weretested with solid-phase RIA versus purified TAG-72 and wereshown to be positive, while being negative for reactivity withpurified CEA (Table 1). All the CC MAbs were reactive (Table1) with biopsy extracts of 4 of 5 colon carcinomas from individual patients. Note that B72.3 was not reactive with extract 820while all the CC MAbs were strongly reactive and that most ofthe CC MAbs were more strongly reactive with extract 770than was MAb B72.3. The differential reactivity of the 28 CCMAbs with biopsy extracts of six different mammary carcinomas is worthy of note. While MAb CC52 was reactive with 4of 5 colon carcinomas, it was negative to all 6 breast carcinomas.In contrast, MAbs CC49 and 92 were reactive with all sixmammary carcinomas. A spectrum of reactivity was observedfor the CC MAbs with the six breast carcinomas: 0 of 6 (n =1), 1 of 6 (n = 1), 2of6(n = 7), 3 of 6 (n = 12), 4 of 6 (n =3), 5 of 6 (n = 2), 6 of 6 (n = 2); B72.3 reacted with 3 of 6. The28 CC MAbs were also tested for reactivity with membrane-enriched biopsy extracts of 32 normal human tissues. Theseincluded liver (n = 4), spleen (n = 5), kidney (n = 2), lung, bonemarrow, stomach, heart muscle, thyroid, phytohemagglutinin-stimulated and nonstimulated lymphocytes, polymorphonuclear leukocytes, normal colon (n = 2), and erythrocytes ofvarious blood groups (n = 11). As can be seen in Table 2, therewas only minimal reactivity or, in the vast majority of cases, nodetectable reactivity of any of the CC MAbs with these extracts.Of course, as with all assays, a lack of reactivity does not definethe tissue as "negative" for TAG-72, but rather as below thelimits of detection of the assay, and "negative" in comparison

with those results obtained with identically prepared extractsof carcinomas (Table 1). Note that the higher values obtainedwith the normal colon extracts must be taken in light of theresults also obtained with the NS-1 negative control sera. Theimmunoperoxidase studies detailed below did, however, revealsome reactivity of some CC MAbs and B72.3 with some cellsin normal colon preparations.

Immunohistochemical Analysis. The 28 CC MAbs and MAbB72.3 were reacted with 5-^m sections of formalin-fixed tissuesusing the avidin-biotin complex immunoperoxidase method.All 28 MAbs reacted with formalin-fixed tissues because theoriginal 99 hybridoma supernatants were tested for reactivityto formalin-fixed sections of carcinoma biopsies and only those

4590




Table 1 Reactivity of CC MAbs in solid-phase RIA with TAG-72 and carcinoma biopsiesThe CC MAbs and B72.3 were reacted with extracts of colon and breast carcinoma biopsies and purified TAG-72 and CEA as described in "Materials and

Methods." Anti-CEA MAb COL-1 was used as control.

ColoncancerMAbCCICC8CC9ecuCC14CC15CC20CC26CC29CC30CC40CC41CC46CC48CC49CC52CC55CC57CC60CC63CC66CC72CC74CC78CC83CC87CC90CC92B72.3NS-ICOL

1TAG-7211,36410,4909,82410,13010,7539,84310,38810,31510,89210.06210,1169.1618,41510,3379,9598,2279,0339,48416,5139,2859,9136,78010,9557,8558,7559,3259,6056,7226,318Neg1,239CEANeg"NegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNeg11,01272610,64010,89311,31010,61410,0149,86110,26410,20910,15810,85011,526NT2,8199,8477,4421,7488,6998,5521,8279,2098,5722,6529,2037,3525,6059,4059,4601,6673,463Neg9,1888538,9349,17810,41110,2138,7779,37210,2138,4579,5549,9399,660NT3,4728.7486.2752.9668,4508,1781,6018,9228,4324,2128.0647,2874,7588,6227,7972,2761,6015145,0968207,0846,5326,8737,3997,4297,2447,1557,3497,3126,6336,9606,1463,0245,0446,3286,4826,9436,5431,9996,8326,4102.3166,8245,1313,4716,9706,4151,889NegNeg5,0677706.2646,8627,4076,6526.8686,4706,8826,4646,3436,7026,6396,2362.2094,2425,5046,0396,6316,6231,5866,8346,9291,5917,5635,0043,0906,4286,2141,7979215795,780781NegNegNegNeg516NegNegNegNegNegNegNTNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNeg5,307251NegNegNegNegNegNegNegNegNegNegNegNegNegNeg1,837NegNegNegNegNegNegNegNegNeg672NegNeg1,525NegNegNeg2696461,5822,7163,0761.6881,2142,1761,500Neg2,1912,5911,5311,1846421,722Neg1,8661,5111,6242,1041,2801,0782,0981,7432,6472,1352,2594,872716NegNTBreast

cancer275475201,6201,651803Neg687643Neg1,2371,138Neg735Neg4,573NegNegNeg2,1711,400NegNeg4,6663,0212,1525347556,1511.659NegNT930512NegNegNegNegNegNeg630NegNegNegNegNegNeg1,569NegNegNeg569NegNegNeg505NegNegNegNeg1,591NegNegNT1030NegNeg972,805,888IgG

isotypeNeg

2,471Neg2,011Neg

2,178NegNeg,431,934Neg

2.105NegNeg,592,822Neg

2,213NegNeg.515626Neg

1,6216,2082,079NegNegNeg573NegNegNegNegNeg2,413NegNegNeg,727,509632,811,600569.804,319,248,930,1871,520

2,035NegNegNT637NegNT2b11111112b11112a1111111111NA2a

Â°Neg, <500 cpm; NT, not tested; NA, not applicable.

Table 2 Reactivity ofCC MAbs in solid-phase RIA with normal tissue biopsies

MAbCCICC8CC9ecuCC14CC15CC20CC26CC29CC30CC40CC41CC46CC48CC49CC52CC55CC57CC60CC63CC66CC72CC74CC78CC83CC87CC90CC92B72.3NS-1Liver(1 =4)Neg"NegNegNeg*NegNegNegNegNeg*Neg*NegNegNegNeg*NegNegNegNegNegNegNegNegNegNegNegNegNegNeg*NegÂ«NegSpleen(1 =5)NegNegNegNegNegNegcNegNegNegNegrNegNegNegNegNegNegNegNegNegNegNegNegNegcNegNegNegNegNegcNegNegKidneyNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNeg-'NegNegLungNegNeg648631627Neg723NegNeg511820NegNeg931817Neg814956Neg7111,0635291,056NegNeg1,9511,3627471,072NegBonemarrowNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegStomachNegNegNegNegNeg506NegNegNegNegNegNegNegNeg594NegNegNegNegNegNegNeg1,117916619Neg553909NegNegMeanmuscleNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNeg709NegNegThyroidNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNeg639NegNegNegNegPhytohemag-

glutinin-stimulated

lymphocytesNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNormallym

phocytesNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegPolymorphe-nuclear leu

kocytesNeg556571555NegNeg647NegNegNeg571Neg527Neg531NegNegNegNeg532NegNegNegNegNegNeg759531NegNegNormal

colon18048081,0551,3147595487141,060839860875584Neg9371,105579563577Neg691684557724737Neg1.0007379575398162Neg619704623654542681714Neg577727NegNeg504608742699NegNeg668528Neg5936595628246641.134587572RBC(1=11)NegNegNegNegNegNegNegNeg"NegNegNegNT*NegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNeg

Â°All extracts listed as negative (Neg) displayed cpm <500. If several extracts were tested, and one or more extract was >500 cpm, it is indicated in "b, c, d, f "

where cpm of each extract are given.* Liver: CC11, 170.297.978. 124;CC29, 134,675,261, 144;CC30. 142,596, 185, 132;CC48, 126,257, 166, 673; CC92, 368, 701, 482, 789.' Spleen: CC15, 124, 162. 139. 626, 157; CC30, 175, 187, 134, 559, 252; CC74, 141. 208, 170, 112, 518; CC92, 405, 519, 428, 199, 400." RBC: CC26, 279, 454, 139, 254, 344, 194, 514. 154, 244, 239, 244.' NT, not tested.7 Kidney: CC92. 374, 712.

4591




that reacted were chosen for further study. Tissues tested werefive colon carcinomas and normal colon, breast, spleen, liver,pancreas, ovary, brain, lung, prostate, heart, stomach, kidney,skin, adrenal, and lymph node tissues. While the carcinomaswere positive to varying degrees, all the cell types of the normaladult tissues were negative with the following exceptions, allexhibiting minimal binding: superficial goblet cells of the colonand stomach (of intestinal metaplasia); glandular epithelium ofthe endocervix; transitional epithelium of the prostate; bronchial epithelium; sebaceous glandular epithelium of the skin;and superficial mucosal cells (goblet cells) of the small intestine.The binding to these cell types usually involved less than 1% ofthe particular cell type staining, but at times reached 5% ofcells staining. In general, all the CC MAbs had patterns ofreactivity for normal tissues very similar or identical to thatshown and previously described for B72.3 (4). The one normalhuman adult tissue that showed strong staining with the CCMAbs (CC49 and CC83 were tested) and with B72.3 wassecretory endometrium; proliferating endometrium and restingendometrium of postmenopausal women, however, were negative for the CC MAbs and for B72.3 as described in detailpreviously (8).

Differential Reactivities of CC MAbs. As described above, the28 CC MAbs listed in Table 1 could be divided into severalgroups on the basis of isotype and reactivity to extracts of breastand colon carcinomas with solid-phase RIA. In an attempt tofurther define differential reactivities among the 28 CC MAbsand select those MAbs with the greater degree of reactivity forcarcinoma, MAb B72.3 and all of the 28 CC MAbs were titratedagainst extracts of the LS-174T cell line (of which B72.3 ispoorly reactive) and the 173 breast carcinoma extracts. Theresults of the nine CC MAbs selected are shown in Fig. 1. Notethey can be categorized into four major patterns: (a) thoseMAbs (CC46 and 92) with a pattern of reactivity similar to thatof B72.3 as shown in Fig. 1; (b) MAb CC49, which is shown toreact as strongly to the LS-174T colon cell line extract as tothe breast cancer extract; (c) MAbs CC83, 15, and 29, withreactivities to the breast cancer extract that are similar to thatof B72.3 but which react much more strongly to the LS-174Textract than B72.3; (d) MAbs CCI 1, 30, and 40, for which thereactivity to the breast cancer extract is much greater than tothe LS-174T colon cancer extract. These results, taken with theresults of reactivity to biopsies of six breast cancer extracts(Table 1), arbitrarily divide the nine CC MAbs chosen forfurther study into at least seven groups on the basis of differential reactivities.

Purified IgG preparations of B72.3 and the nine CC MAbschosen for further study were then compared with solid-phaseRIA for relative reactivities to purified TAG-72 antigen, abiopsy of a gastric carcinoma, a breast carcinoma, and extractsof normal granulocytes, lung, liver, spleen, and kidney. An anti-

CEA MAb (MAb B1.1), which has been shown previously tocross-react with NCA in granulocytes and normal spleen andlung, was used as an additional control. MAb MOPC-21 wasused as a negative control and was shown to be nonreactivewith all extracts. As can be seen in Fig. 2, all the purified IgGpreparations of the CC MAbs show a greater differential reactivity for carcinoma versus normal tissues than did B72.3, andall showed between a 100-fold and 100,000-fold differentialreactivity for carcinoma versus normal tissue biopsies whenpurified IgG preparations were serially diluted.

Western Blotting. MAb B72.3 and MAbs CC11, 15, 29, 30,40, 46, 49, 83, and 92 were analyzed using Western blottingmethodology for reactivity with the TAG-72 antigen. As shown

07

6

Â§5X"i 4

(CC46) (CC 921 (CC 491

(CC83) (CC 151 (CC29)

(CC 11) JCC30) (CC40)

0 1234567 0123456log MAb DILUTION

01 234567

Fig. 1. Differential reactivities of MAb B72.3 and CC MAbs to carcinomaextracts using solid-phase RIAs. Dilutions of tissue culture supernatant fluids ofB72.3 and the CC MAbs were reacted with extracts of carcinomas in solid-phaseRIA as described in "Materials and Methods." B72.3 was reacted with the 173breast cancer biopsy extracts (O) and the extract from the LS-174T coloncarcinoma cell line (D)- The designated CC MAbs were reacted with the 173breast cancer extracts (â€¢)and the extract from the LS-174T colon carcinoma cellline (â€¢).

ng MAb

Fig. 2. Reactivities of purified IgGs of the CC MAbs and MAb B72.3 topurified TAG-72 and carcinoma extracts versus extracts of normal tissues. Purified IgG preparations of the designated CC MAbs and B72.3 were reacted insolid-phase RIAs (as described in "Materials and Methods") with the following:purified TAG-72 (â€¢);extracts of a stomach cancer biopsy (A); breast cancerbiopsy (â€¢);normal lung and normal liver (D); normal spleen and normal kidney(A); and normal granulocytes (O). MAb B1.1 (42) was used as a control since ithas been shown previously to be reactive with CEA and CEA cross-reactiveantigens in several normal human tissues.

4592




in Fig. 3, and as previously described, B72.3 recognizes predominantly a high molecular weight species (M, > IO6) (Fig. 3A,

Lane 1). Upon longer exposure, multiple lower molecularweight species can be recognized with a minor band at M, ~ 3x IO5. As seen in Fig. 3, all the CC MAbs analyzed recognizesimilar molecular weight forms of TAG-72 in the carcinomaextract.

Cell Surface Reactivity. As mentioned previously, TAG-72 isnot well expressed on the surface of established cell lines inculture. One of the few cell lines expressing low levels of TAG-72 is the LS-174T colon carcinoma line. As shown in Table 3,eight of nine CC MAbs chosen for further study showed binding

â€¢â€¢â€¢If

200K-

92 ic

es K-

B 34â€¢fill92K-

68 Kâ€”

Fig. 3. Immunodetection of TAG-72 as recognized by CC MAbs in extractsof LS-174T using Western blotting analyses (see "Materials and Methods"). TheLS-174T tumor xenografts were homogenized using an Omni-Mixer and centri-fuged to remove nuclei and mitochondria. The supernatant was separated bySDS-PAGE on 3-10% polyacrylamide gels. TAG-72 was visualized using Western blotting procedures. Equal amounts of antigen (10 units) were added to alllanes. Monoclonal antibodies used for detection were as follows: Lane l, B72.3;Lane 2, CCI 1;Lane 3, CC15; Lane 4, CC29; Lane 5, CC30; Lane 6, CC40; Lane7, CC46; Lane 8, CC49; Lane 9, CC83; Lane 10, CC92; and Lane 11, MOPC-21. A, 3-h exposure to X-ray film; B, 13.5-h exposure. K, molecular weight inthousands.

Table 3 Cell surface reactivity ofCC MAbsThe CC MAbs and B72.3 were reacted with LS-174T colon carcinoma cells

and nonmalignant WI-38 and/or MRC-5 and Flow 4000 cells as described in"Materials and Methods."

Experiment1CCI5CC30CC49CC92B72.3NS-1Experiment

2ecuCC29CC40CC46CC83B72.3LS-174T3,7201,4404,0802,0501,580Neg8586831,119Neg858NegWI-38Neg"NegNegNegNegNegNegNegNegNegNegNegMRC5

andFlow4000NegNegNegNegNegNegNTNTNTNTNTNT

to the surface of live LS-174T cells in culture and were non-reactive with the surface of the WI-38 or the MRC-5 and Flow4000 control nonneoplastic cell lines. Note that in Experiment2, however, the CC MAbs were generally lower than in Experiment 1 and B72.3 was not reactive to the surface of LS-174Tcells. This antigenic drift of the LS-174T cell line and othercell lines in culture has been detailed previously (23, 24, 49).As shown in Fig. 4, however, when MAb CC49 was analyzedby flow cytometry for reactivity to the surface of freshly isolatedhuman breast carcinoma cells derived from a pleural effusion,both B72.3 and CC49 (but not the MOPC-21 control MAb)react to the surface of the majority of tumor cells, with CC49reacting more strongly than B72.3. A subsequent study (50)demonstrated that six CC MAbs tested targeted human carcinoma xenografts in vivo.

Competition Radioimmunoassays. Nine CC MAbs and B72.3were analyzed as purified IgG preparations in several liquidcompetition RIAs to better define distinctions between bindingsites of these MAbs. As seen in Fig. 5 (A and B) MAbs CC29and 92 competed very poorly in the B72.3 competition RIA.MAbs CC46, 49, and 40 required between 14 and 17 timesmore MAb IgG for competition and competed only approximately 65-75% at highest input levels. MAbs CC11, 15, and30 also competed moderately but could clearly be distinguishedfrom B72.3 in both the amount required for competition (10-to 100-fold more than B72.3) and the extent of maximumcompetition. MAb CC83, on the other hand, competed in amanner similar to that of B72.3.

In the reciprocal MAb CC83 competition RIA (Fig. 6A),however, B72.3 competed very poorly, even at 1000-fold greaterinputs than MAb CC83, thus distinguishing the reactivities ofthese two MAbs. Moreover, MAbs CC30, 46, and 49 couldclearly be distinguished from MAb CC83 in this assay.

Using a MAb CC49 competition RIA, eight of the nine MAbsused as competitors showed only partial or no competition (Fig.5, C and /)); MAb CC83, however, did show complete competition. As shown in the MAb CC83 competition assay, however,these two MAbs could be easily distinguished (Fig. 6/4). Thesedata, taken with the data shown in Fig. 6, B and C using theMAb CC46 and CC30 competition RIAs and previous datashown above, demonstrate that B72.3 and MAbs CCI 1,15, 29,30, 40, 46, 49, 83, and 92 can be distinguished from oneanother.

Affinity Constants. The studies detailed above demonstratethat most of the CC MAbs show greater reactivity to carcinomathan does B72.3 and that each of the CC MAbs appears to beunique. Affinity constants of binding to the TAG-72 antigenwere determined for the purified B72.3 IgG and the purified

' Neg, <500 cpm; NT, not tested.

0 20 40 60 80 100 120 140 160 180 200IMMUNOFLUORESCENCE

Fig. 4. Flow cytometric analyses of binding of MAbs B72.3 and CC49 to thesurface of human breast carcinoma cells isolated from a pleural effusion. Thesolid line represents the sorting pattern with negative control primary antibodyMOPC-21; , pattern of reactivity of MAb B72.3; , MAb CC49reactivity.

4593




20 -

10 -

10' 102 10' 10* 10*

ng COMPETITOR

10' 104

Fig. 5. Competition RIAs using radiolabeled MAb B72.3 IgG (A and B) andradiolabeled MAb CC49 (C and D). Purified IgG preparations of MAbs wereused to compete for binding of the radiolabeled MAb with the LS-174T coloncarcinoma extract as described in "Materials and Methods." Purified IgG from

the MAbs were used as competitors at the indicated quantities. A and C: B72.3(â€¢);CC40 (O); CC49 (â€¢);CC29 (D); CC92 (A); and CC46 (A). B and D: B72.3(â€¢);CC30 (O); CC49 (â€¢);CCI l (D); CCI 5 (A); and CC83 (A).

10' 10' 10' 10* 10 10* 10' 1010' 10' 10*

ng COMPETITOR

Fig. 6. Competition RIAs using purified IgG preparations of radiolabeledMAbs CC83 (A), CC46 (B), and CC30 (C) as described in "Materials andMethods." Purified IgG from the MAbs were used as competitors at the indicated

quantities. CC46 (â€¢);B72.3 (O); CC49 (â€¢);CC30 (D); and CC83 (A).

IgG preparations of nine CC MAbs. As seen in Fig. 7 (opencircles), the Ka of B72.3 was determined to be 2.54 x IO9 M~'.

All the CC MAbs demonstrated Ka values greater than B72.3(Fig. 7) with some as high as 16.18 x IO9 M~' (MAb CC49)and 27.72 x 10" M"1 (MAb CC83). Thus, Ka values could also

be added as a criterion to distinguish these nine CC MAbs fromone another and from B72.3.

CC83Ka = 27 72

--Q.

Ka = 2 54x IO9Â«"'

CC49Ka Â«16 18x 10Â»M-'

CC92Ka - 14.26

ecuKa = 12 60IO'M-'-\Â« x 109M

'"" %"Q-0-

, V ,'^Ãb-

CC30Ka = 8.15x 10"M-'

CC40Ka Â«458

-A_.

CC46Ka = 3.64

0.1 0.2 0.3 0.4 0.5 0.1 0.2 0.3 0.4 0.5

[Bound Abi x IO"9 M

0.1 0.2 0.3 0.4 0.5

Fig. 7. A. determinations of CC MAbs (â€¢)and MAb B72.3 (O) for comparison. The CC MAbs and MAb B72.3 were reacted with purified TAG-72 asdescribed in "Materials and Methods." The ratios of the concentration of bound

to free antibody (Ah) were plotted against the concentrations of bound antibody.The slope represents â€”Ka of each antibody.

- DISCUSSION

MAb B72.3 has been shown to be useful in the study ofhuman carcinoma cell populations (23, 24, 46, 49) and hasdemonstrated potential utility in several different aspects ofcancer management, including use in serum assays, immuno-histological analyses of fine needle aspiration biopsies, immu-nocytological analyses of human serous effusions, and radiolo-calization of human carcinoma lesions //; situ. Our rationalefor the preparation of the second generation CC MAbs wasthat other anti-TAG-72 MAbs could be useful in combinationwith B72.3, or perhaps more useful than B72.3, in those applications.

The original immunogen for the generation of B72.3 was amembrane-enriched fraction of a breast carcinoma metastasisthat had first been passaged through protein A columns toremove endogenous immunoglobulins. Subsequent screening ofhybridoma cultures was done with carcinoma biopsies. Sincewe have shown that the B72.3 rarely reacts with establishedcarcinoma cell lines, it is now obvious that we would not havediscovered this MAb if cell lines were used for either immunogen or screening.

Analyses and purification of the TAG-72 antigen were at firsthampered by the lack of a reproducible source of antigen; celllines were either negative or very poor expressors, and onlylimited quantities of individual human biopsy materials areavailable. The studies of Friedman et al. (49) demonstrated thatfresh human colon biopsies placed in monolayer culture wouldbegin to lose binding of B72.3 within days; this was shown notto be a cell selection phenomenon. It has also been shown thatonly 2 carcinoma cell lines of approximately 40 tested boundB72.3, and this binding was very weak. The studies of HoranHand et al. (23), however, demonstrated the phenomenon thatthree-dimensional spatial configuration had an effect on TAG-72 expression. In monolayer cultures, the very well-differentiated LS-174T cell line was 1 of the 2 lines that bound B72.3weakly. When these cultures were placed in agar or grown asspheroids, the B72.3 binding increased severalfold. When LS-174T cells were grown as xenografts in athymic mice, B72.3binding increased approximately 100-fold and was comparable

4594




to B72.3 binding to many human carcinoma biopsies. Thus, theLS-174T human colon carcinoma xenograft became a sine Quanon for the analysis and utilization of the TAG-72 antigen.

The purification of TAG-72 from LS-174T xenografts usingmultiple purification procedures including B72.3 affinity chro-matography made feasible the production of the second generation anti-TAG-72 MAbs described here. Since a colon carcinoma was used as immunogen in contrast to the original breastcarcinomas used as immunogen for B72.3, the second generation MAbs were given a CC designation.

The selection procedure for the 2567 original hybridomasinvolved reactivity to carcinoma biopsies and lack of reactivityto biopsies of several normal adult tissues. Of the 99 culturesselected for double cloning and further analyses, 28 culturesshowed the desired reactivities to purified TAG-72 and tocarcinoma versus normal adult tissues via both RIAs and inimunohistochemical analyses. All screening was conducted withani imurine IgG reagents, and all 28 CC MAbs were shown tobe IgGs. Nine CC MAbs were selected for further characterization on the basis of their differences of reactivity from B72.3and each other and their ratio of reactivity to carcinoma versusnormal biopsies.

Studies are currently under way to use the CC MAbs togenerate an epitope map of TAG-72. Since it has been extremely difficult to truly deglycosylate the TAG-72 molecule,studies are still being conducted to define the nature of theepitopes recognized by each CC MAb. Analyses of these nineCC MAbs by direct binding assays to a variety of carcinomas,competition RIAs, and Ka measurements have demonstrated,however, that each can clearly be distinguished from one another and B72.3.

It will require extensive investigations to determine if one orseveral of these CC MAbs can be used either as a more efficientsubstitute for, or in combination with, B72.3 in one or severalof the diagnostic and potential therapeutic applications citedabove.

ACKNOWLEDGMENTS

We thank D. Simpson, M. Taylor, K. Siler, K. Irvine, and M.Duberstein for their assistance in these studies.

REFERENCES

1. Colcher, D., lloran Hand. P.. Nuti, M., and Schlom, J. A spectrum ofmonoclonal antibodies reactive with human mammary tumor cells. Proc.Nail. Acad. Sci. USA, 7Â«:3199-3203, 1981.

2. Nuti, M., Teramoto. Y. A., Mariani-Costantini, R., Horan Hand. P., Colcher,D., and Schlom, J. A monoclonal antibody (B72.3) defines patterns ofdistribution of a novel tumor associated antigen in human mammary carcinoma cell populations. Int. J. Cancer, 29: 539-545. 1982.

3. Stramignoni. D.. Bowen. R., Atkinson, M.. and Schlom, J. Differentialreactivity of monoclonal antibodies with human colon adenocarcinomas andadenomas. Int. J. Cancer, 31: 543-552. 1983.

4. Thor, A., Ohuchi, N., Szpak, C. A., Johnston, W. W., and Schlom, J. Thedistribution of oncofetal antigen TAG-72 defined by monoclonal antibodyB72.3. Cancer Res., 46: 3118-3124, 1986.

5. Johnston, W. W., Szpak, C. A., Thor, A., and Schlom, J. Phenotypiccharacterization of lung cancers in fine needle aspiration biopsies usingmonoclonal antibody B72.3. Cancer Res., 46: 6462-6470. 1986.

6. Ohuchi, N., Simpson, J., Colcher, D., and Schlom, J. Complementation ofanli-CEA and anti-TAG-72 monoclonal antibodies in reactivity to humangastric adenocarcinomas. Int. J. Cancer, 40: 726-733, 1987.

7. Thor. A., Gorstein, F., Ohuchi, N., Szpak, C. A., Johnston, W. W., andSchlom, J. Tumor-associated glycoprotein (TAG-72) in ovarian carcinomasdenned by monoclonal antibody B72.3. J. Nati. Cancer Inst., 76: 995-1006,1986.

8. Thor, A., Viglione, M. J., Muraro, R., Ohuchi, N., Schlom, J., and Gorstein,F. Monoclonal antibody B72.3 reactivity with human endometrium: a studyof normal and malignant tissues. Int. J. Gynecol. Pathol., 6: 235-247, 1987.

9. Johnson, V. G., Schlom, J., Palerson, A. J., Bennett, J., Magnani, J. L., andColcher, D. Analysis of a human tumor-associated glycoprotein (TAG-72)

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

identified by a monoclonal antibody B72.3. Cancer Res., 46:850-857, 1986.Arklie, J., Taylor-Papadimitriou, J., Bodmer, W., Egan, M., and Millis, R.Differentiation antigens expressed by epithelial cells in the lactating breastare also detectable in breast cancers. Int. J. Cancer, 28: 23-29, 1981.Milkens, J., Buijs, F., Hilgers, J., Hageman, P., Calafat, J., Sonnenberg, A.,and van der Valk, M. Monoclonal antibodies against human milk-fat globulemembrane detecting differentiation antigens of the gland and its tumors. Int.J. Cancer, 34: 197-206, 1984.Magnani, J. L., Steplewski, /.., Koprowski, II. and Ginsburg, V. Identification of the gastrointestinal and pancreatic cancer-associated antigen detectedby monoclonal antibody 19-9 in the sera of patients as a mucin. Cancer Res.,Â«.â€¢5489-5492,1983.Prat, M., Morra, 1., Bussolati, G., and Comoglio, M. CAR-3, a monoclonalantibody-defined antigen expressed on human carcinomas. Cancer Res., 45:5799-5805, 1985.Ellis, I. O., Hinton, C. P., MacNay, J., Elston, C. W., Robins, A., Owainati,A. A., Blarney. R. W., Baldwin, R. W., and Ferry, B. Immunocytochemicalstaining of breast carcinoma with the monoclonal antibody NCRC 11: a newprognostic indicator. Br. Med. J., 290: 881-883, 1985.Hayes, D. F., Sekine, H., Ohno, T.. Abe, M., Keefe, K., and Kufe, D. W.Use of a murine monoclonal antibody for detection of circulating plasmaDF.1antigen levels in breast cancer patients. J. Clin. Invest., 75: 1671-1678,1985.Burchell, J., Durbin, H., and Taylor-Papadimitriou, J. Complexity of expression of antigenic determinants recognized by monoclonal antibodies HMFG-1 and HMFG-2, in normal and malignant human mammary epithelial cells.J. Immunol., 131: 508-513, 1983.Hilkens, J., Kroezen, V., Bonfrer, J. M. G., DeJong-Bakker, M., and Bruning,P. F. MAM-6 antigen, a new serum marker for breast cancer monitoring.Cancer Res., 46: 2582-2587, 1986.Linsley, P. S., Ochs, V., Laska, S., Horn, D., Ring, D. B., Frankel, A. E.,and Brown, J. Elevated levels of a high molecular weight antigen detected byantibody Wl in sera from breast cancer patients. Cancer Res., 46: 5444-5450. 1986.Sekine, H., Ohno, T., and Kufe, D. Purification and characterization of ahigh molecular weight glycoprotein detectable in human milk and breastcarcinomas. J. Immunol.. 135: 3610-3615. 1985.Burchell, J., Wang, D., and Taylor-Papadimitriou, J. Detection of the tumor-associated antigens recognized by the monoclonal antibodies HMFG-1 and2 in serum from patients with breast cancer. Int. J. Cancer. 34: 763-768,1984.Schlom. J. Basic principles and applications of monoclonal antibodies in themanagement of carcinomas: the Richard and Hinda Rosenthal FoundationAward Lecture. Cancer Res., 46: 3225-3238, 1986.Lan, M. S., Bast, R. C., Colnaghi, M. L, Knapp, R. C., Colcher, D., Schlom,J., and Metzgar, R. S. Co-expression of human cancer-associated epitopeson mucin molecules. Int. J. Cancer, 39: 68-72. 1987.Horan Hand, P., Colcher, D., Salomon. D., Ridge, J., Noguchi, P., andSchlom, J. Influence of spatial configuration of carcinoma cell populationsin the expression of a tumor associated glycoprotein. Cancer Res., 45: 833-840, 1985.Horan Hand, P., Nuti, M., Colcher, D., and Schlom, J. Definition ofantigenic heterogeneity and modulation among human mammary carcinomacell populations using monoclonal antibodies to tumor-associated antigens.Cancer Res., 43: 728-735, 1983.Klug. T. L., Sattler, M. A., Colcher, D., and Schlom, J. Monoclonal antibodyimmunoradiometric assay for an antigenic determinant (Ã‡A72) on a novelpancarcinoma antigen (TAG-72). Int. J. Cancer, 38: 661-669, 1986.Paterson, A. J., Schlom, J., Sears, H. F., Bennett, J., and Colcher, D. Aradioimmunoassay for the detection of a human tumor-associated glycoprotein (TAG-72) using monoclonal antibody B72.3. Int. J. Cancer, 37: 659-666, 1986.Johnston, W. W., Szpak, C. A., Lottich, S. C., Thor, A., and Schlom, J. Useof a monoclonal antibody (B72.3) as an immunocytochemical adjunct todiagnosis of adenocarcinoma in human effusions. Cancer Res., 45: 1894-1900, 1985.Martin, S. E., Moshiri, S., Thor, A., Vilasi, V., Chu, E. W., and Schlom, J.Identification of adenocarcinoma in cytospin preparations of effusions usingmonoclonal antibody B72.3. Am. J. Clin. Pathol., 86: 10-18, 1986.Johnston, W. W., Szpak, C. A., Lottich, S. C., Thor, A., and Schlom, J. Useof a monoclonal antibody (B72.3) as a novel immunohistochemical adjunctfor the diagnosis of carcinomas in fine needle aspiration biopsy specimens.Hum. Pathol., 17: 501-513, 1986.Nuti, M., Mottolese, M., Viora, M., Perrone Donnorso, R., Schlom, J., andNatali, P. G. Use of monoclonal antibodies to human breast tumor associatedantigens in fine needle aspirate cytology. Int. J. Cancer, 37: 493-498, 1986.Szpak, C. A., Johnston, W. W., Roggli, V., Kolbeck, J., Lottich, S. C,Vollmer, R., Thor, A., and Schlom, J. The diagnostic distinction betweenmalignant mesothelioma of the pleura and adenocarcinoma of the lung asdefined by a monoclonal antibody (B72.3). Am. J. Pathol., Â¡22:252-260,1986.Colcher, D., Esteban, J. M., Carrasquillo, J. A., Sugarbaker, P., Reynolds,J. C., Bryant, G., Larson, S. M., and Schlom, J. Quantitative analyses ofselective radiolabeled monoclonal antibody localization in metastatic lesionsof colorÃ©ela!cancer patients. Cancer Res., 47: 1185-1189, 1987.Esteban, J. M., Colcher, D., Sugarbaker, P., Carrasquillo. J. A., Bryant, G.,Thor, A., Reynolds, J. C., Larson, S. M., and Schlom, J. Quantitative andqualitative aspects of radiolocalization in colon cancer patients of intrave-

4595




nously administered MAb B72.3. Int. J. Cancer, 39: 50-59, 1987.34. Renda. A., Salvatore, M., Sava, M., Landi. R., Lastoria, S., Coppola, L.,

Schlom. J., Colcher, I).. and Zannini, G. Immunoscintigraphy in the follow-up of patients operated on for carcinoma of the sigmoid and rectum. Dis.Colon Rectum, 30: 683-686, 1987.

35. Colcher, D.. Esteban, J., Carrasquillo. J. A., Sugarbaker. P., Reynolds, J. C.,Bryant. G.. Larson. S. M., and Schlom. J. Complementation of intracavitaryand intravenous administration of a monoclonal antibody (B72.3) in patientswith carcinoma. Cancer Res., 47: 4218-4224. 1987.

36. Martin, E. W., Mojzisik, C. M., Hinkle. G. H., Sampsel, J., Siddiqi, M. A.,Tuttle. S. E., Sickle-Santanello, B., Colcher. D., Thurston, M. O., Bell. J.G., Farrar, W. B., and Schlom, J. Radioimmunoguided surgery: a newapproach to the intraoperative detection of tumor using monoclonal antibodyB72.3. Am. J. Surg., in press, 1988.

37. Sickle-Santanello, B. J., O'Dwyer, P. J., Mojzisik. C., Tuttle, S. E., Hinkle.

G. H., Rousseau. M., Schlom. J., Colcher. D.. Thurston, M. O.. Nieroda,C.. Sardi. A.. Minton. J. P., and Martin, E. W. Radioimmunoguided surgeryusing the monoclonal antibody B72.3 in colorÃ©ela!tumors. Dis. ColonRectum, 30: 761-764. 1987.

38. Tom. B. H., Rutzky, L. H.. Jakstys, M. M., Oyasu. R., Kaye, C. !.. andKahan. B. D. Human colonie adenocarcinoma cells. I. Establishment anddescription of a new cell line. In Vitro (Rockville). 12: 180-191, 1976.

39. Herzenberg, L. A., Herzenberg, L. A., and Milstein, C. Cell hybrids ofmyelomas with antibody forming cells and T lymphocytes with T cells. In:D. M. Weir (ed.). Handbook of Experimental Immunology, pp. 25.1-25.7.London: Blackwell Scientific Publications. 1978.

40. Colcher. D.. Keenan. A. M.. Larson, S. M., and Schlom, J. Prolonged bindingof a radiolabeled monoclonal antibody (B72.3) used for the in situ radioim-munodetection of human colon carcinoma xenografts. Cancer Res., 44:5744-5751, 1984.

41. Lowry. O. H.. Rosebrough, N. J., Fair, A. L., and Randall, R. J. Proteinmeasurement with the Polin phenol reagent. J. Biol. Chem., 193: 265-275.1951.

42. Colcher, D., Horan Hand. P.. Nuti, M., and Schlom, J. Differential bindingto human mammary and non-mammary tumors of monoclonal antibodiesreactive with carcinoembryonic antigen. Cancer Invest., /: 127-138, 1983.

43. Muraro, R., Wunderlich, D., Thor, A., Lundy, J., Noguchi, P.. Cunningham,R., and Schlom, J. Definition by monoclonal antibodies of a repertoire ofcarcinoembryonic antigen differentially expressed in human colon carcinomas versus normal adult tissues. Cancer Res., 45: 5769-5780, 1985.

44. Potter, M.. and Lieberman, R. Genetic studies of immunoglobulins in mice.Cold Spring Harbor Symp. Quant. Biol.. 32: 187-202. 1967.

45. Potter, M. Immunoglobulin-producing tumors and myeloma proteins ofmice. Physiol. Rev., 52:632-684, 1972.

46. Greiner, J. W., Fisher, P. B., Pestka, S., and Schlom, J. Differential effectsof recombinant human leukocyte interferons on eel! surface antigen expression. Cancer Res., 46:4984-4990, 1986.

47. Heyman, B., Holmquist, G., Borwell, P., and Heyman, U. An enzyme-linkedimmunosorbent assay for measuring anti-sheep erythrocyte antibodies. J.Immunol. Methods, 68: 193-204, 1984.

48. Scatchard, G. The attractions of proteins for small molecules and ions. Ann.NY Acad. Sci., 5/: 660-672. 1949.

49. Friedman. E., Thor, A., Horan Hand, P., and Schlom, J. Surface expressionof tumor-associated antigens in primary cultured human colonie epithelialcells from carcinomas, benign tumors, and normal tissues. Cancer Res., 45:5648-5655, 1985.

50. Colcher, D., Minelli, M. P., Roselli, M.. Muraro, R., Simpson-Milenic, D.,and Schlom, J. Radioimmunolocalization of human carcinoma xenograftswith B72.3 second generation monoclonal antibodies. Cancer Res., 48:4591-4603, 1988.

4596



1988;48:4588-4596. Cancer Res Raffaella Muraro, Masahide Kuroki, David Wunderlich, et al. Glycoprotein 72 AntigenMonoclonal Antibodies Reactive with the Tumor-associated Generation and Characterization of B72.3 Second Generation

Updated version

http://cancerres.aacrjournals.org/content/48/16/4588

Access the most recent version of this article at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/48/16/4588To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]



Documents

Generation and Characterization of B72.3 - Cancer Research